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Anti-vax and Immunity From Cognitive Dissonance

Anti-vax and Immunity From Cognitive Dissonance

I spend entirely too much time on Twitter reading and participating in arguments with anti-vaxers. This is not to convince them they’re wrong so much as to convince fence-sitters and lurkers they’re wrong. Because they are. Their arguments go around in circles so fast, you could get whiplash trying to follow them. I’m going to try to compose a post that follows some order despite the subject matter being so disordered.

Anti-vaxers can’t decide what’s wrong with vaccines

First there was Wakefield…and the argument that the MMR vaccine caused autism. It’s been widely debunked by many large, well-designed studies, but that means nothing to the anti-vax community. So little that they went looking for something that the researchers had missed, like thimerosal. Which wasn’t in the MMR, and which has been also thoroughly tested, and whose removal from all childhood vaccines besides flu had no effect on the rates of autism diagnosis. Anti-vaxers like to call it mercury and equate this ethyl mercury with methyl mercury or even elemental mercury so that the miniscule amount that’s in a vaccine that you can get without it is so neurotoxic that getting a single flu vaccine is even more dangerous than getting all the vaccines before thimerosal was removed from them.

Well, something has to cause autism, right? So it’s the aluminum adjuvants! You can explain all you want about the ubiquity of aluminum in the environment, the amount in our brains that we tolerate just fine that far exceeds the amount in a vaccine, the fact that there is more aluminum in what we eat and breathe every day than in a full course of vaccines over years, or that the only people who’ve ever shown deleterious effects from aluminum have had renal failure or renal failure plus parenteral nutrition.

And again, they misrepresent aluminum adjuvants as elemental aluminum. The adjuvants are a compound that’s designed to stimulate an immune response and then get flushed out by the kidneys in short order. Anti-vaxers’ arguments frequently depend on a deliberate misunderstanding of chemical compounding. One I butted heads with frequently declared “pro-vaxers think aluminum is salt!” when presented with both the explanation of how the adjuvant is an aluminum salt, not elemental aluminum, but also with the example of how dangerous sodium is on its own, but how safe it is when combined with a chloride to make table salt. You can’t make this stuff up. They want to take the aluminum hydroxide out of the vaccines, but that means using more of the antigen or using a live virus instead of a weakened or killed one…so clearly if we did that, the vaccines would be even less safe, so we shouldn’t have them at all! You can’t win here.

They’ll move the goalposts over to formaldehyde. As with ethyl mercury and aluminum hydroxide, no amount of telling them about the dose making the poison will have an impact. And as with aluminum, no amount of comparing natural levels of exposure will convince them that they survive worse than vaccines. The amount of formaldehyde inn our bodies is already several thousand times higher than what’s in a vaccine, and is absolutely vital for cell reproduction. As well, the most dangerous way to be exposed to formaldehyde is through the lungs. Some anti-vaxers will reiterate the injection vs. ingestion trope here, forgetting entirely that vaccines aren’t inhaled. Tell them that baby’s going to breathe in a ton of formaldehyde from the new clothes and sheets and stuffed toys than he’s going to get from a vaccine, and you’ll get all kinds of crazy responses. Or blocked.

Now, when it’s actually penetrated to one or two of them that these individual ingredients are not toxic or not toxic in the amounts given in vaccines, they will often turn to “synchronous toxicity,” which means that it’s THE COMBINATION of all of these at once that creates a devastating neurotoxin. At this point, they will probably have told you that since there are no studies of the individual ingredients vs. saline (the only placebo they will accept) that we need to look into synchronous toxicity. Forget that the vaccines are tested for this every time they’re not tested vs. saline. Or that we have decades of clinical data to show that VACCINES ARE SAFE AND DON’T CAUSE AUTISM. I suppose we’ll have to devise tests on every possible combination of ingredients using saline as a placebo until we get the results the anti-vaxers want.

Anti-vaxers will read only poorly designed studies

They will also misinterpret good studies and cite them as proof of their anti-vax claims, not knowing that the studies actually contradict them.

Their favorite studies on ethyl mercury are the ones that say “since we didn’t have information on the toxicity of ethyl mercury, we substituted the numbers for methyl mercury.” If you give them the actual information on ethyl mercury, they’ll find a reason to dismiss it or ignore it entirely. Or block you.

They still insist that the studies that seemed to indicate higher levels of aluminum in the brains of Alzheimer’s patients (currently not considered supportive of causation) are clearly also supportive of less than a microgram of aluminum in a vaccine causing ASD. They will play math games that make it look like all the vaccines are given at once so as to compare it to a single day of exposure from food, or they’ll insist that “injection is different from ingestion” as if that causes the aluminum in a vaccine to multiply from less than a microgram to several milligrams. A study comparing different kinds of aluminum adjuvants using cells in a petri dish becomes concrete evidence that all adjuvants immediately cause irreversible brain damage.

While they bang the drum of “injected vs. ingested,” they’ll dig up information on the results of exposure to high levels of inhaled formaldehyde and insist that the trace amounts of formaldehyde will, guaranteed, produce equivalent results. They can’t explain, though, why morticians and scientists who work with preserved large specimens, or people who work with textiles don’t develop autism.

As I said above regarding “synchronous toxicity,” you can provide them with long lists of studies of the individual ingredients, combinations of ingredients, individual vaccines, combination vaccines, and they simply will refuse to read them. Some dismiss them out of hand because of some perceived conflict of interest (this came from the CDC! CORRUPT!!) while others will read only the title and make all their assumptions from there. Sometimes they’ll peek at the abstract, select a sentence or a few words that they can interpret as admission of danger or doubt, screenshot it and share it as if this proves their point. On rare occasions, they’ll skim through the study with the same motivation as they had for the abstract, picking a fragment that has no impact on the data, but that they think changes everything. What they will not do is read anything critically or learn enough science to understand what constitutes a good study.

They pretend that they like diseases better than vaccines

I say this because liking the diseases involves a fair amount of mental gymnastics to paint the diseases as benign. If you tell them that worldwide, over 130,000 people died from measles, they will dutifully explain that those are only people in third world countries (they’re not) or with compromised immune systems (also wrong) or “poor health,” which boils down to victim blaming for not eating what they’re supposed to or jogging every day or whatever. Don’t ask an anti-vaxer to define “health.” You’ll never get out of that rabbit hole.

Playing with statistics is one of their favorite ways to downplay the danger of disease. They don’t want worldwide stats, they want the number of people who died in an area with high vaccine compliance during certain years. They don’t care about morbidity, only mortality, and don’t want to see outcomes based on numbers of cases if numbers based on total population better suit their narrative. They point to relatives who survived a given disease as proof that disease is harmless, and refuse to acknowledge that there are still plenty of people who lived through the epidemics and remember the people who didn’t.

One of the weirder things they argue is the dangers of “shedding.” They claim to want their kids to get “natural immunity” to preventable disease by getting the diseases themselves. They never quite explain how natural immunity to rabies or tetanus is going to help their children survive them the next time, though. Anyway, in order to prove how frightened they are of vaccines and “vaccine injury,” they support the idea that the recently vaxed will shed a disease simply by being in the same room as their precious unvaxed children. Now, shedding is possible, but only in two vaccines – rotavirus and oral polio (not given in the US anymore.) In order for these to shed, you need direct contact with urine or feces. People who get a reaction to a live virus vaccine can theoretically “shed” a weakened form of the disease, but try finding verified cases of that (I found one on PubMed after a good amount of searching.)

So here it is that they want their kids to get diseases that have serious consequences, but are terrified that they might be exposed (somehow) to a weakened form that won’t actually cause the disease. At the pediatrician’s office, they could catch measles from an infected child who left a couple of hours ago, but they’re scared of the baby that just got the MMR.

To further reinforce their fears, they have increased the number of conditions that constitute “vaccine injury” to the point that pretty much any disease or disorder is caused by vaccines no matter that it predates vaccines or has a known cause or hasn’t increased except by population increases. They also increase figures for conditions that they say are caused by vaccines (like the diseases themselves) by adding unrelated diagnoses to things like polio while also saying that the reason we see drops in VPDs in areas with high vaccine compliance is because doctors are diagnosing VPDs as other diseases to cover up the “fact” that vaccines don’t work. They are, again, terrified that their children will catch these diseases (the ones being vaccinated against, or the ones they imagine the vaccines cause) while still espousing the idea that catching these diseases is a good thing for their kids’ immune systems.

Just keep arguing

As pointless as it is to argue with people who have developed this amazing ability to resolve cognitive dissonance by ignoring it, it’s not pointless to argue with them. What you see in these discussions is this lunatic fringe, but others are reading. If those others are not entirely convinced of your point by your sharing of factual information and scientific support, they’ll quite possibly be turned away by the contorted, angry, constantly self-contradictory arguments of the anti-vaxers with whom you’re engaged. Let them dig their own holes. Keep calm and keep bringing on the science. Herd Immunity depends on it.

Wednesday Links

Wednesday Links

Image courtesy of Science Blogs

A recent MIT study said that glyphosate caused nearly every disease known to man. Except it wasn’t an MIT study at all.

A researcher discusses harassment by animal rights activists and explains why animal research is needed (and how he treats his animals) in Defending Animal Research

Food is not magic, and superfoods do not prevent disease.

Vaccines are safe, according to an analysis of 67 independent papers. We know this because it’s been covered in newspapers and magazines in print and online. Here’s the paper itself.

Along the vaccine lines, it didn’t take long for the conversation at USA Today to turn to Miracle Mineral Solution (aka Miracle Mineral Supplement or just MMS) being a cure for autism. Because, of course, vaccines cause autism. (How do vaccines cause autism?) In case you don’t know, this is a solution that misguided people give their autistic children orally or rectally (the same people who complain about the trauma of getting a needle are giving their autistic kids frequent, regular enemas. . .) because they think it’s going to “fix” them.

But this stuff is industrial strength bleach, which is used to treat water that won’t be used for drinking, and to strip textiles. The FDA warns people to throw it out if they have it. Advocates of alt-med and “natural solutions” even warn you away from it – Johnathan Campbell, who believes food is medicine, does not pull any punches explaining how and why it’s dangerous. Signs of the Times, a site that’s entirely woo-friendly, has nothing good to say about it, either. Health Wyze, otherwise supportive of alternative medicine, calls it a Fraud.

So it’s not only science-based sites that decry this stuff. The Guardian warns people away, Science-Based Medicine explains why it is dangerous woo, The Thinking Person’s Guide to Autism considers this stuff even more appalling than chelation and chemical castration., and Thinking is Dangerous explains the chemistry behind MMS. James Randi Foundation informs us that if this stuff isn’t scary enough for you, you can buy MMS2, which is essentially pool shock.

Liz Ditz provides a long list of links from science sites and bloggers telling about the dangers of MMS. PLoS has some additional links.

If all this doesn’t scare you, have this lovely video:

Wednesday Links

Wednesday Links


The environmental benefits of genetically modified crops is explored in Conservation Tillage, Herbicide Use, and Genetically Engineered Crops in the United States: The Case of Soybeans

A piece on the claim that GMOs are under-studied, With 2000+ global studies affirming safety, GM foods among most analyzed subjects in science pretty much demonstrates that no, they are not.

Neonicotinoid pesticides are sprayed on crops, and they are bad for good insects. But they’re good for selling plants. Engineered pest resistance doesn’t get sprayed and affects only pests that attack the specific crops. Just sayin’.

Organic foods may have been sprayed with pesticides, too – and isn’t necessarily any better for you. Being free of GMOs makes no difference.


A friend and I were blocked from commenting on an online discussion on the terrible, horrible things that are vaccines. This is a typical technique of anti-vaxxers. A detailed description of what it means to be anti-vaccine is on Science-Based Medicine It’s from 2010, but classics never get old.

Because of a new study analyzing the actual risks of vaccination (hint – nearly none, even less compared with disease outcomes) the pro-vaccine message is finally getting the press it deserves. USA Today, The Daily Beast, Think Progress (I know, not a big anti-vaxxer magnet) The New York Times and Time. Even The Economist reminds us that we should take our medical advice from science, not celebrities.


Viruses may be responsible for several cancers. The Big Idea That Might Beat Cancer and Cut Health-Care Costs by 80 Percent explores a virus that may trigger certain kinds. Vaccination to prevent cancer might work better than treating it after the fact, ya think?

Quadruple amputee soldier learns to adapt to life with transplanted arms.

‘Molecular movies’ will enable extraordinary gains in bioimaging, health research


This is stupid, which means it made me laugh a lot.

Wednesday Links

Wednesday Links

reality check


In the wake of pretty much every outbreak of every vaccine-preventable disease, comments on the news articles fill up with people who still think that vaccines cause autism. One article keeps getting referred to, “22 Studies that Prove Vaccines Cause Autism.” I’m not going to link, it doesn’t need any more hits, because it already shows up on the first page of many searches on vaccines. Instead, I’m going to direct you to Liz Ditz’s excellent rebuttal.

Foodbabe proves over and over that she’s all style and no substance. The Foodentists dissect her attack on Lean Cuisine and the Grocery Manufacturers Association with many facts about GMOs that she apparently doesn’t know – or chooses to ignore.

On the topic of GMOs, Gilles-Eric Séralini’s paper linking glyphosate to tumors in rats, which was retracted last year because of methodological and statistical flaws, has been re-published in a journal with apparently less exacting standards. I’m thinking along the lines of “repeat a lie often enough and it becomes the truth.”

SFARI tells us that autism is not the only neurodevelopmental disorder that’s on the rise. The numbers may actually be a good thing, because it means that more people are getting needed treatment.

You know that study that said watching porn shrinks your brain? Well, maybe not so much. Christian Jarrett at Wired talks about the study’s many shortcomings.

Business Insider has an interesting piece on the Myers-Briggs personality test. By the way, I’m ENFP.

Sometimes things are partly true, or true but misrepresented. In those cases, we don’t need debunking, we need. . .

Critical Thinking

I got a little gut-punch here, because I hate neuroscience hype, but I also did a few little happy dances reading about optogenetics. I pick on optogenetics, but… and Moving on from optogenetic frustrations are actually not too far from the mark, though. I think it is possible to get excited about a new method without looking at it as a be-all and end-all breakthrough. . .as long as you look at the research and stay away from the media version.

Another thing that gets oversold is brain imaging. Again, cool, but not as magical as it’s portrayed sometimes. Lots of times. Virginia Hughes talks realistically about the limits and potential of neuroimaging.

A longread (28 pages) on critical thinking. I have to admit, it’s still open in another tab as I write this. Written from a legal viewpoint, as in how something would stand up in court when exposed to scrutiny, but relevant in a general sense as well.

I often take issue with people who are strict “nurturists” because they are so unspecific about what “environment” is and what it does. Genetics and epigenetics are mechanisms that are, while still being incompletely understood, more logical and straightforward than the more nebulous claims of environmental influence. Many of the people I’ve run across take a Lamarckian viewpoint, or even imagine evolution as a personal change (more akin to Pokemon evolution than anything we see in biology!) So I read Developmental Plasticity and the “Hard-Wired” Problem all the way through, and was pleasantly surprised to see a thoughtful and detailed approach to the “Nature vs. Nurture” question. I don’t know how convinced I am, but it’s more than I’ve been by anyone else presenting this argument.


If you wish to make a gene from scratch explains that, well, it’s not really as easy as that.

Cath Ennis explains how epigenetics works in two parts.

Video – Pallas Cat kittens

Somehow not as freaky when they’re kittens, and funny to see domestic cat behavior in response to the intrusion of the camera.

What has been happening. . .

What has been happening. . .

And why I’ve been away so much. This started about three weeks ago, when my Mom went in to see the oncologist for her biopsy results, and the staff didn’t think that her shortness of breath and loss of consciousness was simply stress. They sent her to the emergency room, and I got a call that if I didn’t come to pick up my Dad, he’d be picked up by a long-term care facility and they might not release him to my Mom because she was so sick. Talk about incentive. I hadn’t finished doing laundry, so I went down with whatever I had to wear, my medications, and an aerobed.

Turns out, she’d been having these problems for a while (and, of course, downplayed them so nobody would be concerned) and it was a pulmonary embolism. A small one, but I don’t think size really matters much. Thank goodness they insisted. But here’s where the fun begins. You see, Dad has been declining mentally for several years, and his condition is another thing Mom has been downplaying. The reasons are numerous and complex, and I’m not going to get into too much detail because that’s outside of this narrative. Since Mom’s got atrial fibrillation and has been on blood thinners, which she stopped so she could get her biopsy (Non-Hodgkin’s lymphoma, treatable with chemo) it became kind of complex to get her clotting factor right at the same time as they broke up the clot in her lung – and while she was there, they wanted to install a chemo port and give her her first treatment.

This meant over a week in the hospital for her.

This meant over a week of caring for my Dad by myself.

At this point, my new meds had not kicked in, I was still having panic attacks, and I was trying to process a whole bunch of information without the benefit of Adderall, either. And my Dad’s dementia is. . .bad. Looking at the description of a seven-stage progression, he’s between five and six, and awfully close to entirely stage six. After just a couple of days with him, taking him back and forth to the hospital and then being yelled at later for not having told him Mom was in the hospital, not being able to do anything except have the same conversations with him over and over without him getting upset that I wasn’t engaging him, being awakened at all hours of the night and early in the morning either because he was wandering the house (sometimes on his way outdoors) or waking me up to ask where Mom was, I was really on edge. I texted my sister and asked if she could relieve me for the weekend, and, bless her heart, she showed up on Thursday night. Not only did it help me out enormously, but I now had someone else to corroborate my story about his condition.

Things were relatively OK, but then Mom called me on a Saturday to tell me that Dad was in the hospital – this was his second time, but his first one (a couple of weeks prior to Mom’s for the embolism) she didn’t tell me anything until he was home. This time, she really shouldn’t have been driving, but she would have if we hadn’t come down, so hubby and I headed down on Sunday to take her to visit Dad. We got her a wheelchair to take her around the hospital, because she needed it. Dad was really out of it, sleeping slouched in a chair when we arrived, so we went and got lunch. When we came back, nothing had changed, and Mom found that she couldn’t wake him up, that he muttered a few incoherent things, and we realized that his arms and legs were ice cold.

When we called the nurse and they realized that they couldn’t get his blood pressure, we were shooed out of the room, and soon there were more doctors and nurses than could even fit. They moved him to the bed, and tried to get a chest x-ray, but he was uncooperative and physically fighting them off. They figured that in addition to the one infection he had, he probably also had pneumonia, so they began an IV drip of antibiotics for that. He was conscious when we finally got back into the room, but nothing he said made any sense.

Once we knew he was out of danger (because we were pretty worried for a while) we brought Mom home. I called her the next day, and she had spoken to him, and he was doing better, but still thought he was being held in a jail for something. She had no idea if she actually had someone to give her a ride to her second chemo appointment, so I figured I would drive her myself and then go to the hospital to see if they could just care for him for a couple more days – because even this had not been enough for her to actually get a home health aide or a visiting nurse. I knew that there was no way that she could care for him and keep him out of danger while recovering from an infusion.

So this is where it gets dramatic.

I slept badly, of course, and set off a little before 6:30AM to pick her up. I went in with her to speak with the oncologist and we discussed, frankly, the reality that she would not be able to care for Dad by herself, and I think that hearing it from the doctor lent it credibility that it didn’t have coming from me. She agreed that as much as she wanted Dad to be able to stay at home with her, it was in her best interest that he be cared for around the clock somewhere else for at least a little while when she was feeling like crap.

I got Mom settled in with her pillow and blanket and book, and headed out to the hospital. I went to the nurses’ station, trying to keep out of his sight so I could speak to them without upsetting him. They shocked the heck out of me by announcing that they had been calling all morning because it was time to release him! I explained that this was really, really, really bad timing, because my Mom would be hooked up to chemo drips until at least 4PM, and I couldn’t take him to the oncologist’s office OR leave him home alone. Well, they told me, the papers had already been signed, so I’d have to take him and do one or the other. No room for negotiating.

The social worker was at the desk, and I asked her if he could stay. No. Are there any short-term places he can go? No, he doesn’t qualify for short-term rehab, so he’d have to go into long-term care, and then he’d be there permanently. I didn’t want to place him somewhere permanently, and I especially didn’t want to be the one responsible for placing him permanently, against my Mom’s wishes. I asked if I could speak to the doctor who signed the release. (n.b., at this point, my Dad has not seen me, but he no longer has a guard in the room – he’s all by himself, seated on a pad that sounds an alarm every time he gets up, at which point, nurses rush in and make him sit down again.)

The doctor comes out, and I have to say, I have not been treated so condescendingly or disrespectfully by a doctor in close to 20 years. I’m not naming the hospital or the doctor – I’m going to write to them, I don’t need them to have a bunch of people descending angrily upon them, because my anger should be just about all they can handle! I tried to explain to him that Mom was getting chemo all day, I live an hour and a half drive away, Dad’s dementia makes it impossible for her to care for him while she’s dealing with her own treatments, and isn’t there some way he can just keep Dad there for even one more day? He gives me the same line about either I take him home, or he gets committed permanently and there’s no way he’ll ever come home, and he’s a professional gerontologist and I should know that Dad’s mental condition will decline rapidly if he goes into a home and I’ll be responsible for giving him a death sentence.

That’s when I threw out the names of the other doctors with whom I had consulted who agreed that he needed nursing care while mom was sick (GP as well) and suddenly he’s all “Oh, I know them. Good doctors. Well, I wish you luck,” and then walked away with a smile as if he had not just implied that I don’t care if my father dies in a nursing home.

Believing I had no choice and needing to do something quickly, because Dad was beginning to get really angry with the alarms and the nurses and such, I arranged for a liaison from the home closest to my parents’ house to start the admission process. Dad had seen me at this point, so I had to sit with him for a bit, but I needed to call Mom and it’s impossible to make a phone call with Dad there because he gets upset if you’re talking but not to him. Of course, he tried to follow me out, and the alarm went off, and the nurses came, and he was fighting and yelling.

Down the hall, I tried Mom’s cell phone, but she didn’t have it or it wasn’t on, so I called the main number for the oncologist. The receptionist passed the message, and shortly after, I was talking to Mom about what I had been forced to do and why – then she passed the phone to one of the nurses and the oncologist’s social worker. They were pretty furious, because I’d been lied to. Yes, my only option was a nursing home, but it wasn’t a prison. We could take him out any time.

Trying to explain this to my Dad was an awful experience. He didn’t get the concept – of anything. He forgot who I was. He was angry because he’d been kept alone in this room for so long and didn’t understand why this alarm kept going off and why nobody would let him walk around. He was tired of waiting around to go visit whomever he thought he was visiting, because he didn’t remember that he was the patient. When the rep from the nursing home arrived, she was wonderful. It was obvious that she understood how to handle people with memory issues, and had the patience of a saint. She figured out the one thing that caught my Dad’s full attention – he wanted to take care of Mom. She told him that he was going to need to build up his strength so he could do that, so he was going to stay in this place and do physical therapy every day until he was ready to be Mom’s caregiver. And when she saw that I would tell him what was happening and then he’d get mad because nobody had told him this was happening, over and over again, she told me that the hospital social worker needed to see me so I should say goodbye to him. It got me out of the room, and Dad accepted it – but nobody actually wanted to talk to me. She just knew that was the only way to disengage.

I went back and stayed with Mom until her chemo was done. I hadn’t had anything to eat, so I was given some crackers and coffee by the oncology nurse. When the chemo was done, I went out to pick up Mom’s prescriptions while she packed clothes for Dad. I got back, and she was on the phone with Dad, explaining to him that he needed to get strong so he could take care of her. The moment they hung up, my sister called. I may have been a bit abrupt (sorry, Jen!) but I had been up since 4:30, had eaten nothing but those crackers since 6AM, and still had to drive to the home and drop off the clothes and pick up dinner from the diner.

The home was nicer than some, not as nice as others, but the staff was good, and nobody was restrained. Dad wanted me to take him on a tour, and this was just not an option at this point. Fortunately, a staffer was approaching us, and I asked her if she could show him around. She agreed, and I said my goodbyes. Picked up food. Ate. Drove home, got there about 10:30.

A couple of days later, I called Mom. She’s talked with Dad, and he thinks this place is pretty luxe, and he’s doing physical therapy so he can come home. She, meanwhile, has been able to sleep whenever she needs to and for as long as she wants, and get sick without Dad trying to “help” her. She didn’t want him to be taken away from home, but for the moment, things seem to be working out, and that takes a huge weight off my mind.

Learning from Research, The Results.

Learning from Research, The Results.

This is the part where my brain is going to explode. I might need to break this up into more than one post.

Preparation of HMECs

A single HMEC in its log phase was plated, and expanded to 1.4 × 106 to 1.5 × 106 cells (Fig.1). Plating efficiency during the two transfers of plates was 67 ± 0.9(mean ± SE)%. Based on these values, the number of cells that should have been produced at the time of harvest was calculated as 3.2 × 106(1.4 × 106/0.67/0.67). This value predicted that each cell harvested underwent 21.6 generations from the initial single cell. Doubling time was 48 h.

Strategy of cell culture. A single HMEC was inoculated in a well by limiting dilution, and the cell was expanded up to approximately 106 cells. Based on the plating efficiencies during the two transfers and the actual final cell count, the number of cells that should have been produced at the time of harvest and the number of generations observed were calculated. DNA was extracted from the final cells, and used for bisulfite sequencing. Six independent cultures were performed.

HMEC – Human Mammary Epithelial Cells. They were put into a container, allowed to reproduce, and then they were checked to see if the right number of cells were made after specific numbers of generations. There were six containers of these cells. Once enough generations had passed and there were enough cells, their DNA was tested with the bisulfate test (illustrated in my earlier post.)

Gene Selection and Their Expression Levels

Methylation statuses were determined by bisulfite sequencing for CGIs in the promoter regions of the E-cadherin,p41-Arc, SIM2, 3-OST-2, and Cyclophilin A genes; CGIs in the downstream exon/introns of theE-cadherin, p41-Arc, and SIM2 genes; CpG sites outside CGIs of the E-cadherin and p41-Arcgenes; a NM-CGI of the MAGE-A3 gene; and differentially methylated region (DMR) of the H19 gene (Fig.2A). The former five genes were selected because they had CGIs in the downstream exon/introns that met a strict criterion of CGIs, regions of DNA of >500 bp with a G+C ⋝ 55%, and observed CpG/expected CpG of 0.65 (Takai and Jones 2002). The MAGE-A3 gene and the DMR of the H19 gene were selected as a representative NM-CGI and a region critically involved in genomic imprinting, respectively. By quantitative RT-PCR analysis, their expression levels were shown to range from almost none (SIM2 and MAGE-A3) to very high (E-cadherin), with p41-Arc, 3-OST-2 andCyclophilin A being intermediate (Fig. 2B).

Structures and expressions of the genes analyzed. (A) Schematic representation of the genomic regions analyzed. Regions analyzed by bisulfite sequencing are shown by closed boxes, and designations A–L correspond to panels in Fig. 3. CGI-P: a CGI in the promoter regions; CGI-outside: a CGI outside the promoter regions; Non-CGI: CpG sites outside CGIs; and DMR: differentially methylated region. All panels are drawn to the same scale. (B) Expression levels of the seven genes in HMECs.
Genome Res. 2003 May 13(5) 868-74, Figure 2

Sorry, I can’t even. All I know from this is that they looked at the results of the bisulfite sequencing and found what they were looking for – the methylation status in the CpG Islands from promoter regions of DNA stayed almost exactly the same. Unmethylated CGIs from non-promoter regions were more likely to become methylated. I’m afraid I don’t have the ability to explain this to you or tell how accurate or flawed it may be. I’m taking the researchers’ word on it. Correct me if I’m wrong.

Establishment of How to Measure MPERs

The CGI in the promoter region of the E-cadherin gene (Fig.3A), the non-CGI region of thep41-Arc gene (Fig. 3F), the CGI in the promoter region of theMAGE-A3 gene (Fig. 3K), and the DMR of the H19 gene (Fig. 3L) were found to contain two major populations of clones. The two major populations were considered to represent the methylation pattern of the two alleles in the original single cell. The methylation patterns of the two major populations were different from each other in the six cultures, which indicated that the HMECs before cloning had diverse patterns of methylation, but the patterns were relatively conserved during the culture from a single cell to approximately 106 cells. Therefore, we measured the number of errors in the methylation pattern based upon the culture from a single cell to approximately 106 cells. An MPER of a region in a culture was calculated from the number of errors in methylation pattern as described in Methods, and an average MPER of the region was calculated from the six MPERs obtained for the six cultures.

MPERS – Mammalian Protein Extraction Reagent
AlleleAn allele is one of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. If the two alleles are the same, the individual is homozygous for that gene. If the alleles are different, the individual is heterozygous. Though the term “allele” was originally used to describe variation among genes, it now also refers to variation among non-coding DNA sequences.

So after making all those cells, they looked to see where and whether methylation status had changed.

Distribution of unmethylated and methylated CpG sites shown by bisulfite sequencing. Unmethylated and methylated CpG sites are shown by open and closed circles, respectively. (A)–(C) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the E-cadherin gene. (D)-(F) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the p41-Arcgene. (G), (H) A CGI in the promoter region and a CGI outside the promoter region of the SIM2 gene. (I) A CGI in the promoter region of the 3-OST-2 gene. (J) A CGI in the promoter region of the Cyclophilin A gene. (K) A CGI in the promoter region of the MAGE-A3 gene, which is normally methylated. (L) A CGI in the differentially methylated region of the H19 gene.

Here’s where they found the differences:

Genome Res. 2003 May 13(5) 868-74, Figure 3

To examine the effect of an arbitrary selection of the “original methylation pattern” in ambiguous cases, a permutation test was performed for the CGI in the E-cadherin promoter region of HMEC10. One of the clones #5–#14 (Fig. 3A) was hypothesized as one of the original methylation pattern, and the number of errors in the methylation pattern was calculated. The numbers ranged from 18–22, and these values were expected to result in the average MPER ranging from 0.022–0.023. Similar permutation tests were performed for the CGI in exon 2 of the E-cadherin gene of HMEC12 and HMEC15. The numbers of errors in methylation pattern ranged from 13–16 for HMEC12 and from 12–15 for HMEC15, and these values were expected to result in the average MPER ranging from 0.050–0.058. These showed that arbitrary selection of the original methylation pattern in ambiguous cases does not seriously affect the resultant average MPER.

Some changes weren’t so cut and dried, so they checked those cases and found that they weren’t significant enough to change the findings.

The efficiency of bisulfite conversion was examined by analyzing DNA with no methylation in the CGIs in the promoter region and exon 2 of the E-cadherin gene. In the CGI in the promoter region, none of the 600 cytosines at CpG sites (30 CpG sites per clone, 20 clones analyzed) remained unconverted, showing that unconversion rate was almost 0 in this region under our experimental condition. In the CGI in exon 2, one of 483 cytosines at CpG sites (23 CpG sites per clone, 21 clones analyzed) remained unconverted, showing that the unconversion rate was 0.0021. These values showed that the MPERs in CGIs in the promoter regions are 10-fold more than the unconversion rates.

The bisulfate conversion was also tested separately for control to make sure the results would be valid in the experiment. This reinforced the finding that the promoter regions stayed stable.

MPERs and Fidelities of Methylation Pattern in the Genome

The average MPERs obtained for each region are summarized in Table1. Unmethylated CGIs in the promoter regions showed MPERs between 0.018 and 0.032 errors/site/21.6 generations. In contrast, CGIs outside promoter regions showed significantly higher MPERs, ranging from 0.037 to 0.091 (P < 0.01 or 0.005). MPERs in the CGIs outside the promoter regions were more than twice as high as those in the promoter regions of the same genes. MPERs in Various Genomic Regions

NM-CGI of the MAGE-A3 gene and methylated alleles of the DMR of the H19 gene showed MPERs of 0.002 and 0.007, respectively. Any genomic regions that were normally methylated, whether or not they were in CGIs, showed significantly lower MPERs than those unmethylated. This was particularly clear when the MPER of the allele methylated at DMR of the H19 gene was compared with that of the other unmethylated allele.

Interpretation of the tables, summary of findings.

This is not as good as part one, sorry. In other news, I couldn’t watch Besharam because it sucked, so I didn’t learn any Hindi, either. One more post to go in this series. Anyone who can clarify/explain better than I can, please comment – I’d appreciate it.

Learning from Research, Slowly and Methodically.

Learning from Research, Slowly and Methodically.

I was given a challenge on Twitter, and some people dismissed me as a failure because I didn’t have the academic background to come back with a quick answer. (I also discovered that I knew the answer, but forgot the words because of post-surgical anomia. I digress.) I find that this is a problem with a lot of people with certain types of expertise. They forget what it was like back when they were first learning, and no longer have the patience to explain. I don’t think it helps that there is a shit-ton of people on the internet spouting nonsense and being taken seriously. Naturally, some of them will assume that I’m doing the same, but I really don’t want to be lumped in with them, so I’m going to show them the process I go through, and how seriously I take learning new things and separating fact from fiction.

As I said in my previous post, you guys are wicked smart, and I am very often in awe of how much you know. But one thing you’re not so good at is communicating to people outside your fields of expertise. This is why we have bad science journalism. Ask Ed Yong. However, if you want to stop all your discoveries from degenerating into misrepresentation or woo, then you need people who can translate Science into English.

I was given a long, information-dense study, Fidelity of the Methylation Pattern and Its Variation in the Genome by Malcolm M. Campbell, so it’s going to take several posts to dissect, research, learn the background information, and try to explain it in an accessible way. I fully expect to be wrong several times, and encourage people to correct me – in such a way that ordinary people can “get it.” So here goes:


The methylated or unmethylated status of a CpG site is copied faithfully from parental DNA to daughter DNA, and functions as a cellular memory. However, no information is available for the fidelity of methylation pattern in unmethylated CpG islands (CGIs) or its variation in the genome. Here, we determined the methylation status of each CpG site on each DNA molecule obtained from clonal populations of normal human mammary epithelial cells.

Methylation turns genes or pieces of genes “on” or “off”. There’s a detailed explanation of various ways it does this in the components of the whole process from DNA to cell, but it’s kind of hard to understand if you haven’t done a lot of reading beforehand. I’ll give you the link anyway.

CpG sites – the quick and dirty Wikipedia definition is this: The CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. If you don’t remember from your Biology classes, or your biology classes never taught you, your entire DNA strand consists of combinations of four nucleotides – Cytosine, Guanine, Taurine, and Adenosine. I’m not going to get into that right now, because it’s just going to confound this with too much information, but if you think about the movie “Gattaca,” you’ll notice those four letters. In a movie about genetic engineering. Because those are the four letters you see in an illustration of a piece of DNA. The researchers were looking at the parts where the cytosine and guanine were next to each other.

Specifically, they were looking at epithelial cells from normal breast tissue. The link may be a little difficult to understand, but I think if you read all the way through, you’ll at least understand some of the reasons these cells were chosen. They have a lot of unique characteristics, and they’re pretty tough.

So the idea here is that we already know that if the cytosine and guanine pair are methylated in the on position or the off position in the DNA, that they’re going to stay that way in the cells that are produced by those instructions from the DNA. What we don’t know is that if that pair is unmethylated, will the cells made from the DNA instructions also be unmethylated? IOW, if they’re not already told to be switched on or told to be switched off, will they still be in that “neutral” position? In order to test that, they took a bunch of those epithelial cells and tested each one to see if it was methylated or unmethylated so they could get them to reproduce and see what happened.

This illustration is not specific to this piece of research, but keep reading, and you’ll see how it relates.. I wanted to give you a visual aid in case you learn better that way.

Methylation pattern error rates (MPERs) were calculated based upon the deviation from the methylation patterns that should be obtained if the cells had 100% fidelity in replicating the methylation pattern. Unmethylated CGIs in the promoter regions of five genes showed MPERs of 0.018–0.032 errors/site/21.6 generations, and the fidelity of methylation pattern was calculated as 99.85%–99.92%/site/generation. In contrast, unmethylated CGIs outside the promoter regions showed MPERs more than twice as high (P < 0.01). Methylated regions, including a CGI in theMAGE-A3 promoter and DMR of the H19 gene, showed much lower MPERs than unmethylated CGIs. These showed that errors in methylation pattern were mainly due to de novo methylations in unmethylated regions. The differential MPERs even among unmethylated CGIs indicated that a promoter-specific protection mechanism(s) from de novo methylation was present.

This explains how they figured a reasonable range of variation. The “islands” of unmethylated cytosine/guanine pairs in five genes over 21.6 generations (this is statistics, not absolute numbers. You clone enough cells, you sure as heck can get six tenths of a generation.) stayed unmethylated most of the time. This came from promoter regions, which are the areas in DNA that call the shots. It’s more likely that instructions from promoter regions are going to be followed.

The unmethylated cells that didn’t come from promoter regions showed more deviations – the cells after several generations were twice as likely to be different from the originals than the ones that came from the promoter regions. The methylated cells, which, as I mentioned, already have the specific instructions to turn a gene on or off, were more likely to maintain their integrity even if they weren’t from promoter regions. The unmethylated cells didn’t’ have that instruction, and hadn’t been told to stay unmethylated (because they weren’t from promoter regions) and so they just did whatever seemed right at the time and, well, mistakes were made.

CpG methylation is known to serve as cellular memory, and is involved in various biological processes, such as tissue-specific gene expression, genomic imprinting, and X chromosome inactivation (Jones and Takai 2001; Bird 2002; Futscher et al. 2002;Strichman-Almashanu et al. 2002). These important functions of methylations are based upon the fact that the methylated or unmethylated status of a CpG site is faithfully inherited. The methylated status of a CpG site is inherited upon DNA replication by the function of maintenance methylase, represented by DNA methyltransferase 1, which is located at replication forks and methylates hemimethylated CpG sites into fully methylated CpG sites (Leonhardt et al. 1992; Araujo et al. 1998; Hsu et al. 1999). The unmethylated status of a CpG site is inherited by not being methylated upon DNA replication or any other occasions. Unmethylated CpG sites generally cluster to form a CpG island (CGI), and most CGIs are kept unmethylated (Gardiner-Garden and Frommer 1987; Bird 2002). Methylations of CGIs in promoter regions are known to cause transcriptional silencing of their downstream genes by changing chromatin structures and blocking transcription initiation (Bird 2002;Richards and Elgin 2002). There are limited numbers of CGIs that are normally methylated (normally methylated CpG islands; NM-CGIs) (De Smet et al. 1999; Futscher et al. 2002). CpG sites outside CGIs, especially those in repetitive sequences, are also normally methylated (Bird 2002).

CpG methylation is important. It is carried on pretty faithfully when cells reproduce. It’s also important that unmethylated CpG remains unmethylated, and that’s usually passed on to new cells as well. Most of the unmethylated sites form a cluster called a CpG Island, or CGI. If these unmethylated CGIs become methylated, then it changes what genetic instructions get turned on or off in future generations of cells, if they’re in promoter regions. But it’s not always bad for CGIs to be methylated, because sometimes that’s on purpose.

I’m going to hold off on the transcription and chromatin stuff for later, because I think it’ll stick better when the paper goes into more detail.

To keep the methylation pattern, maintenance of both methylated and unmethylated statuses of CpG sites during DNA replication is necessary. However, the fidelity of the methylation pattern has been analyzed only for the maintenance of the methylated status (Wigler et al. 1981; Otto and Walbot 1990; Pfeifer et al. 1990). The fidelity in maintaining the methylated status of an exogenously introduced DNA was shown to be 94% per generation per site by Southern blot analysis (Wigler et al. 1981). The fidelity in maintaining the methylated status of a CGI in the 5′ region of the PGK1 gene, which was derived from the inactive X chromosome, was estimated to be 98.8%–99.9% per site per generation by the ligation-mediated PCR method after chemical cleavage of DNA (Pfeifer et al. 1990).

We’ve already studied methylated CpG sites and found that it’s pretty consistent. Some studies attesting to that are cited. We know that keeping them unmethylated is also important, but that hasn’t been investigated to our satisfaction.

Normally unmethylated regions might show different fidelities from normally methylated regions. Even among the unmethylated CGIs, the fidelities of their methylation pattern have been suggested to be different according to their location against a gene promoter. Methylation of CGIs in promoter regions almost always leads to transcriptional silencing while that of CGIs outside promoter regions does not (Gonzalgo et al. 1998; Jones 1999). Considering the cellular expense in maintaining methylation pattern, a cell could sacrifice the fidelity of methylation pattern for CGIs outside promoter regions. In addition, by recent genomic scanning techniques for methylation changes (Ushijima et al. 1997; Toyota et al. 1999; Costello et al. 2000; Jones and Baylin 2002), aberrant methylations of CGIs in cancers are observed in a nonrandom manner (Toyota et al. 1999; Costello et al. 2000; Kaneda et al. 2002a; Kaneda et al. 2002b). It is indicated that CGIs outside promoter regions were more frequently methylated than those in promoter regions (Nguyen et al. 2001; Takai et al. 2001; Kaneda et al. 2002a; Asada et al. 2003).

Unmethylated CGIs are more likely to change than methylated ones. Unmethylated CGIs from promoter regions of the DNA pretty consistently shut down the things they’re supposed to shut down, exactly as planned. Unmethylated CGIs from outside promoter regions of the DNA are not so good at that – they’re more likely to become methylated when they’re supposed to stay unmethylated. Some of this methylation of unmethylated CGIs has been seen in cancer. So that’s one example of why we don’t want this to happen.

Here, we analyzed the methylation status of each CpG site on each DNA molecule by the bisulfite sequencing technique (Clark et al. 1994) in six clonal populations of normal human mammary epithelial cells (HMECs), for CGIs in the promoter regions, CGIs outside the promoter regions, and CpG sites outside CGIs. By analyzing the deviation from the most common two patterns, MPERs, which reflected the fidelity in replicating both methylated and unmethylated statuses, were measured.

Like a five-paragraph essay here. Restating what they’re going to do and how they’re going to do it. Remember the illustration? Bisulfite sequencing technique. (Really detailed explanation, Wikipedia explanation).

And now my brain is very, very tired. I am going to watch “Besharam” because I’m also trying to learn Hindi, and I might as well be looking at Ranbir Kapoor while I’m doing it. Heh. I will continue this in a later post. Feedback is welcome and encouraged.

Science People!

Science People!

I’ve been getting a lot of attention on Twitter for the last couple of posts, and that’s given me a lot of articles to read, blogs to keep up with, and Twitter users to follow. Some people got a little testy, and I don’t blame them, because they know more than I do. I get it.

Let me tell you something right now. I am not a professional scientist. I got my Bachelor’s degree in Spanish Language and Literature back in the early 80s, and distanced myself from science since I had to take my only B-track class in all of High School in Biology. I didn’t get it, I didn’t see the point, I put no effort in, and I sucked at it.

That’s ADHD.

But then I started reading books about the brain, and that struck a chord with me because my brain is not the nice neurotypical model. I started reading blogs and websites about the brain, and medicine, and genetics. I learned how to read published research (and occasionally got friends who would sneak me links to full text articles) and would search in the middle of searches when I found terms I didn’t understand or biological processes or mechanisms that were new to me but essential to understanding what I was reading.

This obsessive pursuit of information is also ADHD, BTW.

This means that there are gaps in my knowledge. I am not ashamed to admit that you know more than I do. Please don’t get angry with me when I’m wrong – explain to me why I’m wrong and then tell me how to understand it the right way. I don’t want to be right to win arguments or lord it over people, I want to be right because I have the correct information. You can help me with that.

Thing is, one thing I know I’m really good at is teaching other people things. I take my mistakes, the process by which I figured something out, and the way it works at the most basic level, and try to use that to explain what I know in a way so that other people can “get it.” There are several college students out there pursuing degrees in science because I got them all excited about it. They’re getting the chance I missed out on.

So, you want more minions? (MUHAHAHAHA!!) Give me comments. Help me understand. Because if you help me understand, I can help other people understand. I’m an intelligent woman, I’ll get it pretty quickly, and when I don’t, I’m not in the least ashamed to admit that I was wrong. We can have a mutually supportive and respectful interchange, and I’ll do my part to explain things in an accessible way, using the tools you give me.

Really. Comment. email. Bring it on. I love you guys!

Your Inner Fish

Your Inner Fish

I loved this book, and now PBS is making a miniseries with Neil Shubin. I can’t wait.

A long time ago, right after I read it, I put up a series of posts on a forum detailing the wonderful things I had learned from it. After a while, the threads were hijacked by people who just didn’t get it – or didn’t want to get it – and they disappeared into obscurity. But I stand by what I wrote, and now that this book is back in public view, I want to share these thoughts again. This is a long read, over 4,000 words, and it’s taken from a forum thread, so there are parts that don’t flow entirely well, but I don’t want to edit or rewrite it because it captures the wonder and excitement I felt when I first read the book and I don’t want to change that.

So settle down with a nice cup of tea if you’re ready to go below the fold.

Read the rest of this entry

10 Things I Have Learned About Abortion from Pro-lifers.

10 Things I Have Learned About Abortion from Pro-lifers.

1. Women choose to have sex. Men are apparently not involved in this decision-making process.

2. Women who do not use birth control are irresponsible and should never have sex.

3. Women who use birth control are also irresponsible, because they know that birth control is not 100% foolproof and should never have sex.

4. Being pro-life has absolutely nothing to do with religion. It’s just a coincidence that my God is opposed to abortion, and if yours isn’t, then you’re worshiping the wrong God.

5. No matter how many examples you find of God-sanctioned infanticide in the Bible, it in no way indicates that God is OK with baby-killing. Baby-killing on his orders is OK because reasons. If he says it’s OK, it’s OK, but he definitely didn’t say abortion was OK except in the parts where he did.

6. All the aborted babies could have gone on to do great things. None of the aborted babies would have been “welfare queens” or criminals or deranged genocidal dictators.

7. People are lined up to adopt babies. If you give your baby up for adoption, it will find a loving family. It definitely, positively, won’t join the half million kids already available for adoption or be one of the 23,000 who age out of the system without being adopted every year. Oh, and it will be happy with its family, who will never turn out to be abusive in any way.

8. It is never OK to abort a baby that resulted from consensual sex. Conception circumstances are paramount, which is why it’s OK to abort rape babies. Consensual sex babies are alive at the moment of conception because of consent. Rape babies are alive at the moment of conception, too, but it’s OK to abort them because they aren’t the consequences of the choice of an irresponsible woman. Don’t ask me to explain this, I’ve tried and tried and still don’t get it.

9. If abortions are illegal, nobody will need them. Only 1% of all abortions are for high-risk situations like the life of the mother or significant defects in the fetus, and letting women die and having babies who are severely handicapped (even if they’re guaranteed to die after birth) is a risk that people who aren’t dealing with these situations are willing to accept.

10. Even if you are too poor to support a child, too young to be a parent, too ill mentally or physically to be a parent, addicted to drugs and unemployed and homeless, married to an abusive spouse or a pedophile, the baby is a gift from God and all your problems will go away as long as you don’t get an abortion.